An Efficient Solid Phase Extraction Method for Purification and Analysis of Compound-Specific Plant Sugar Stable Hydrogen Isotope Values.

RATIONALE: The stable hydrogen isotope composition (δ2H) of plant compounds can serve as environmental or metabolic proxies, but interpretations are hindered by insufficient mechanistic understanding. This can be improved by analyzing the δ2H values of metabolic intermediates, such as sucrose, which is the direct substrate of cellulose and a main transport sugar. However, current preparation methods for carbohydrates in general and sucrose in particular are not time-efficient.

METHODS

We evaluated methods that use acetylation of soluble carbohydrate plant extracts to aid in purification as well as isotopic analysis of plant sugars such as sucrose. Extracts were obtained using either hot water or hot 80% ethanol. Acetylated extracts were then purified using an established liquid-liquid separation (LL) method or a new solid phase extraction (SPE) method that we developed. We also evaluated glucose produced from starch after enzymatic digestion. Method performance was evaluated based on quantified yields and the impact on measured δ2H values.

RESULTS

Acetylated sucrose and starch-derived glucose were sufficiently resolved for gas chromatography in all cases. No isotopic biasing was detected for any method. Acetylated sucrose yields differed among methods, with 80% ethanol resulting in approximately threefold higher extraction yield compared to water, and SPE giving smaller but still sufficient yields compared to LL. Sample throughput was doubled with the SPE method compared to the LL method, which allows for larger batch sizes compared to LL.

CONCLUSIONS

We developed an efficient method to analyze compound-specific plant carbohydrate δ2H values using gas chromatography-isotope ratio mass spectrometry (GC-IRMS). This can be applied in experiments aimed at investigating the processes that shape cellulose δ2H values, including deconvoluting the metabolic and hydro-climatic sources of isotopic variation.